Báo cáo Y học: Cloning and characterization of novel snake venom proteins that block smooth muscle contraction pptx

Cloning and characterization of novel snake venom proteinsthat block smooth muscle contraction Yasuo Yamazaki1, Hisashi Koike1, Yusuke Sugiyama1, Kazuko Motoyoshi1, Taeko Wada1, Shigeru

Cloning and characterization of novel snake venom proteins

that block smooth muscle contraction

Yasuo Yamazaki1, Hisashi Koike1, Yusuke Sugiyama1, Kazuko Motoyoshi1, Taeko Wada1,

Shigeru Hishinuma2, Mitsuo Mita2and Takashi Morita1

Departments of1Biochemistry; and2Pharmacodynamics, Meiji Pharmaceutical University, Tokyo, Japan

In this study, we isolated a 25-kDa novel snake venom

protein, designated ablomin, from the venom of the

Japan-ese Mamushi snake (Agkistrodon blomhoffi) The amino-acid

sequence of this protein was determined by peptide

sequencing and cDNA cloning The deduced sequence

showed high similarity to helothermine from the Mexican

beaded lizard (Heloderma horridum horridum), which blocks

voltage-gated calcium and potassium channels, and

ryano-dine receptors Ablomin blocked contraction of rat tail

arterial smooth muscle elicited by high K+-induced

depolarization in the 0.1–1 lM range, but did not block

caffeine-stimulated contraction Furthermore, we isolated three other proteins from snake venoms that are homolog-ous to ablomin and cloned the corresponding cDNAs Two

of these homologous proteins, triflin and latisemin, also inhibited high K+-induced contraction of the artery These results indicate that several snake venoms contain novel proteins with neurotoxin-like activity

Keywords: snake venom; neurotoxin; helothermine; cysteine-rich secretory proteins; ablomin

Over the past 30 years, a plethora of toxins have been

isolated from poisonous organisms, such as snakes,

scorpi-ons, spiders, and micro-organisms These natural toxins use

a variety of approaches to arrest the homeostatic

mecha-nisms of other living orgamecha-nisms, including disruption of

intracellular signal transduction and cytoskeleton

organiza-tion [1–4], and activaorganiza-tion or inhibiorganiza-tion of blood coagulaorganiza-tion

factors [5–10] Toxins that block synaptic transmission,

called neurotoxins, are widely distributed in venoms These

toxins include the conotoxins from cone snails, agatoxins

from spiders, and scorpion toxins [11–16] These toxins exert

their potentially lethal effects by specifically and potently

blocking a variety of ion channels, including those that

conduct Na+, K+, and Ca2+ Therefore, neurotoxins have

been employed as useful tools to investigate the structure

and function of these ion channels [17–20] A large number

of neurotoxin families have also been found in the venom of

Elapidae snakes These toxins, the a-neurotoxins [21]

(represented by a-bungarotoxin [22,23], a-cobratoxin

[24–27], and erabutoxin [28,29])potently and specifically

prevent nicotinic acetylcholine receptor activation A second

family of snake venom neurotoxins, the dendrotoxins, are

homologous to Kunitz-type serine protease inhibitors and act primarily by blocking neuronal K+channels [30,31] In contrast to the neurotoxin-rich venom from Elapidae snakes, the venom from other deadly snakes, including Viperidae and Colubridae snakes, contain surprisingly few neurotoxins, although some neurotoxic phospholipases have been discovered [32–36]

In this report, we describe the isolation of a novel protein, ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi, a member of the Viperidae family) When applied to arterial smooth muscle preparations from rat-tails, ablomin blocks K+-stimulated contraction This effect is similar to that resulting from application of calciseptine, a well-characterized neurotoxin from black mamba (Dendroaspis polylepis polylepis) Calciseptine is a known blocker of L-type Ca2+channels, a property that underlies its ability to block K+-induced contractions of aortic smooth muscle and spontaneous contractions of uterine smooth muscle [37] Furthermore, we demonstrate that several snake venoms contain ablomin-like proteins, which may constitute a novel venom protein family

E X P E R I M E N T A L P R O C E D U R E S

Materials The lyophilized venom of A blomhoffi was a kind gift from

S Iwanaga (The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan)[38] Other snake venoms and venom glands were purchased from the Japan Snake Institute (Gunma, Japan) Superdex 75 pg and 200 pg, SP–Sepharose High Performance, and Q-Sepharose Fast Flow columns were from Amersham–Pharmacia Biotech The Vydac Protein & Peptide C18 HPLC column and the COSMOSIL 5C18 AR-300 HPLC column were the products of JASCO (Tokyo, Japan)and Nacalai Tesque (Kyoto, Japan), respectively Endoprotease Lys-C was

Correspondence to T Morita, Department of Biochemistry,

Meiji Pharmaceutical University, 2-522-1, Noshio, Kiyose,

Tokyo 204-8588, Japan,

Fax/Tel.: + 81 424 95 8479,

E-mail: tmorita@my-pharm.ac.jp

Abbreviations: CRISP, cysteine-rich secretory protein; HLTX,

helothermine; PsTx, pseudechetoxin; CAP, CRISPs Antigen 5

proteins, and Pathogenesis-related proteins.

Note: the nucleotide sequences reported here have been submitted to

GenBank database (tigrin, AY093955; ablomin, AF384218; triflin,

AF384219; latisemin, AF384220).

(Received 21 December 2001, revised 12 April 2002,

accepted 18 April 2002)

purchased from Seikagaku Corporation (Tokyo, Japan).

Other chemicals were of analytical grade (Sigma–Aldrich,

Amersham–Pharmacia Biotech., Wako Pure Chemical Ind

and Kanto Chemical Co.)

Purification of proteins

Tigrin was isolated from the extract of Duvernoy’s glands of

Rhabdophis tigrinus tigrinus Ten Duvernoy’s glands were

broken into small pieces after freezing in liquid nitrogen,

and then extracted in 30 mL of 50 mMTris/HCl pH 8.0 for

4 h at 4C After ultracentrifugation, the supernatant was

applied onto Q-Sepharose Fast Flow column (1.6· 10 cm)

in the same buffer, and eluted with a linear gradient from 0

to 0.5MNaCl A major peak eluted at 0.05MNaCl, which

was subsequently purified by chromatography on Superdex

200 pg (2.6· 60 cm column)

Ablomin was purified by three successive

chromato-graphic steps Five hundred milligrams of lyophilized

A blomhoffi venom was dissolved in 3 mL of 20 mM

imidazole/HCl pH 6.8 containing 0.2M NaCl, and

insol-uble materials were removed by centrifugation and

filtration (0.22 lm) The filtrate was loaded onto a

Superdex 75 pg column (2.6· 60 cm)and eluted with

the same buffer The ablomin fractions from two gel

filtration runs (a total of 1000 mg of snake venom)were

pooled and dialyzed against 50 mMTris/HCl, pH 8.0, and

applied to the Q-Sepahrose Fast Flow column

(1.6· 15 cm) The column was eluted with a linear

gradient of NaCl from 0 to 0.4M at a flow rate of

2 mLÆmin)1 Chromatographic fractions containing

ablomin were then dialyzed against 20 mM

imidazole-HCl, pH 6.0, and fractionated on a SP–Sepharose High

Performance column (1.6· 11 cm) This column was

developed with a linear gradient of NaCl in the imidazole

buffer (0–0.4M, 2 mLÆmin)1)

For the purification of triflin, 500 mg of the venom of

Trimeresurus flavoviridiswas applied to the SP–Sepharose

Fast Flow column (1.6· 30 cm)with 10 mM phosphate

buffer, pH 6.8, and eluted with a linear gradient from 0 to

0.15M NaCl, as described previously [39] Fractions

con-taining triflin were detected by Western blotting using

anti-tigrin serum These fractions were pooled and fractionated

on Superdex 75 pg (2.6· 60 cm)in a 50-mM Tris/HCl,

pH 8.0, containing 0.2MNaCl Finally, triflin was purified

by chromatography on a Blue-Sepharose Fast Flow column

(1.6· 15.5 cm)in 50 mM Tris/HCl, pH 8.0, which was

eluted with a linear gradient from 0 to 0.5MNaCl

For purification of latisemin, 500 mg of the venom of

Laticauda semifasciata was loaded onto Superdex 75 pg

(2.6· 60 cm)in a buffer containing 50 mM Tris/HCl,

pH 8.0, and 0.2M NaCl The latisemin fractions were

loaded onto the SP–Sepharose Fast Flow column

(1.6· 11 cm)in 10 mMphosphate buffer, pH 6.8,

contain-ing 0.05MNaCl, and eluted with a linear gradient to 0.2M

NaCl The latisemin fractions were re-chromatographed on

a Mono S column (0.5· 1 cm) in 10 mMphosphate buffer,

pH 6.0, with a linear gradient to 0.2M NaCl, and on

Heparin-Sepharose CL-6B columns (1.6· 14 cm)with

50 mM Tris/HCl, pH 8.0, using a linear gradient from 0

to 0.3MNaCl

All purification steps were performed at 4C with an

FPLC system (Amersham–Pharmacia Biotech)

Amino-acid sequence analysis Proteins were reduced for 3 h at room temperature with

20 mM dithiothreitol in the presence of 0.5M Tris/HCl,

pH 8.5, 6Mguanidine hydrochloride, and 2 mMEDTA in

a volume of 0.5 mL Three microliters of 4-vinylpyridine were then added, and alkylation was allowed to proceed for 3 h at room temperature The S-pyridylethylated proteins were separated from the reagents by C18 reverse-phase HPLC, and the amino-acid sequence was determined by sequencing the peptides obtained by diges-tion with endoprotease Lys-C All the samples were analyzed on Applied Biosystems protein sequencers (mod-els 473 A and 477)

cDNA cloning of proteins The cDNAs encoding tigrin, ablomin, and latisemin were obtained using the RT-PCR method Typically, venom gland total RNA was isolated from the venom gland with ISOGENTM (Wako Pure Chemical Industries, Japan) according to the manufacturer’s protocol 5¢ and 3¢ RACE were carried out to determine the nucleotide sequence of the 5¢ and 3¢ end cDNAs with the SMARTTMRACE cDNA amplification kit (Clontech) The amino-acid sequences of peptides derived from purified proteins were used to design degenerate primers For the first amplification of tigrin and latisemin cDNA, degenerate primers were used for both sense and antisense primers For ablomin cDNA, PCR was performed with single degenerate primer (sense or antisense) and an primer recognizing an adaptor sequence that had been attached to the 5¢ or 3¢ end of cDNAs In the case of triflin, PCR was carried out using habu cDNA library as a template [40] with a degenerate primer and an adaptor primer The PCR products were subcloned into the pGEM T-easy vector (tigrin and triflin)or pUC19 vector (ablomin and latisemin)and sequenced with the DSQ 2000 L DNA sequencer (Shimadzu, Japan) Primers used this study are described as follows: tigrin, sense 5¢-AA(C,T)GT(A,C,G,T) GA(C,T)TT(C,T)AA(C,T)TC(A,C,G,T)GA(A,G)TC-3¢ (corresponding to amino acids 1–8 in tigrin)and antisense 5¢-(A,G)TT(A,G)CA(A,G)TT(A,G)TT(A,G)TA(A,G)TC (A,G)TC-3¢ (corresponding to amino acids 187–193 in tigrin); ablomin, sense 5¢-GGCCATTA(C,T)ACTCAG(A,G) T(A,G)G-3¢ (corresponding to amino acids 114–120 in ablomin)and antisense 5¢-C(C,T)A(C,T)CTGAGT(A,G) TAATGGCC-3¢ (corresponding to amino acids 114–120

in ablomin); triflin, antisense 5¢-GC(A,G)TG(A,G,T)AT (A,G,T)AT(A,G)TC(A,C,G,T)GTCCA-3¢ (corresponding

to amino acids 86–91 in triflin); latisemin, sense 5¢-GA (A,G)AA(C,T)CA(A,G)AA(A,G)GA(A,G)AT(A,C,T)G-3¢ (corresponding to amino acids 11–17 in latisemin)and antisense 5¢-G(A,G)CA(A,G)TT(A,C,G,T)GT(A,G)AA (C,T)TC-3¢ (corresponding to amino acids 183–189 in latisemin)

Contraction measurements on rat-tail arterial smooth muscle

Helical strips of endothelium-free rat-tail arterial smooth muscle were prepared as described previously [41] All the contraction experiments were carried out at room tempera-ture, and all buffers were pre-oxygenated with 100% O

The strips were held at 75 mg resting tension in Hepes/

Tyrode (H-T)solution (137 mMNaCl/2.7 mMKCl/1.8 mM

CaCl2/1 mMMgCl2/5.6 mMglucose/10 mMHepes, pH 7.4)

for 45 min Then, the strips were treated with H-T solutions

containing 1 lM prazosin, to block the effect of

norepi-nephrine via a1 adrenergic receptors, for 30 min The strips

were then exposed to 60 mMKCl H-T solution for 15 min

KCl H-T solution was prepared by replacing the NaCl in

H-T solution with equimolar KCl After washing with

calcium-free H-T solution for 5 min, the smooth muscle

strips were stimulated with 20 mM caffeine H-T solution

For measuring the effect of the proteins, all the H-T

solutions contained the indicated concentrations of

pro-teins

R E S U L T S

Identification, isolation and cloning of tigrin

and ablomin

During the isolation process of a prothrombin

activa-tor from the Duvernoy’s gland of Yamakagashi snake

(R tigrinus tigrinus), we identified a large quantity of a

single chain 30-kDa protein (Fig 1), which we named tigrin

To permit further study, tigrin was purified two

chroma-tographic steps The extract from Duvernoy’s glands was

first separated by anion-exchange chromatography

(Fig 1A), and then the major peak was purified by gel

filtration (Fig 1B) An amino-acid sequence was

deter-mined by peptide sequencing and partial cloning, revealing

that tigrin was structurally homologous to helothermine

(HLTX; 49.0% identity, Fig 1C)from the venom of the

Mexican beaded lizard (Heloderma horridum horridum)

HLTX is known to alter a variety of ion channel acti-vities, including voltage-gated K+channels, voltage-gated

Ca2+channels, and ryanodine receptors [42–44] Because

we speculated that HLTX-like proteins would be wide-spread in snake venoms, we generated an rabbit anti-tigrin serum We then screened several snake venoms with the anti-tigrin serum using Western blotting or ELISAs As a result, we detected immunoreactive proteins in the venoms from three snakes: A blomhoffi, T flavoviridis, and Laticauda semifasciata The immunoreactive proteins were then puri-fied by column chromatography, using anti-tigrin serum as

a detection reagent

Using this procedure, we isolated a novel snake venom protein (named ablomin)from the venom of the Mamushi snake (A blomhoffi)through three purification steps First, the crude venom of A blomhoffi was separated by gel filtration on a column of Superdex 75 pg (Fig 2A) Fractions containing ablomin were identified using with SDS/PAGE, based upon an Mr that was initially deter-mined by Western blotting (Fig 2B) These fractions were further separated by anion-exchange chromatography on Q-Sepharose Fast Flow column (Fig 2C) Ablomin was eluted at the concentration of 0.2–0.3M NaCl (bold line

in Fig 2C) This fraction was then subjected to cation-exchange chromatography on SP–Sepharose High Per-formance (Fig 2D) The purified ablomin migrated with a

Mr of 26 kDa on SDS/PAGE under nonreducing condi-tions and 29.7 kDa under reducing condicondi-tions (Fig 2D, inset) From this purification, we obtained 7 mg of purified ablomin from 1 g of crude venom The N-terminal and partial amino-acid sequences of this protein were deter-mined by peptide sequencing of enzymatically digested peptides (underlined in Fig 3) Based on the obtained partial amino-acid sequence, we cloned ablomin cDNA from the venom gland of A blomhoffi by RT-PCR using degenerate primers The cloned ablomin cDNA was 1336 base pairs in length, encoding a 19-residue putative signal peptide, starting at nucleotide 66, and a 221-residue mature protein (molecular mass 24 932 Da), starting at nucleotide 123 (Fig 3) As expected, ablomin was quite homologous to HLTX, with 52.8% of the deduced amino acids identical to the corresponding residues in HLTX (Fig 4)

The effects of tigrin and ablomin on rat tail arterial contraction

We examined the effects of ablomin and tigrin on high

K+- or caffeine-induced contraction using helical strips of endothelium-free rat-tail arterial smooth muscle Ablomin remarkably inhibited contraction evoked by treatment with high K+, but not that evoked by treatment with caffeine (Fig 5A) In contrast, tigrin did not affect both contraction evoked by either treatment (Fig 6B) The block of contraction by ablomin was concentration-dependent to 1 lM (Fig 5B)and completely reversible after a 45-min washout of protein (data not shown) Inhibition by ablomion was reduced at a concentration of

3 lM (Fig 5B) High K+-treatment of the artery induces membrane depolarization and activates voltage-gated channels, leading to smooth muscle contraction [45,46]

In contrast, caffeine exposure causes transient contraction

by activating ryanodine receptors of the sarcoplasmic

Fig 1 Isolation of tigrin from the Duvernoy’s glands of R tigrinus

tigrinus (A)The extract from Duvernoy’s glands of R tigrinus tigrinus

was fractionated on a Q-Sepharose Fast Flow column with a linear

gradient of NaCl (dotted line) (B)Major peak (bar in A)from

Q-Sepharose Fast Flow column was fractionated by gel filtration on a

Superdex 200 pg column The pooled fraction (bar)contained purified

tigrin (inset, SDS/PAGE; R, reducing conditions; NR, nonreducing

conditions) (C) Primary structure of tigrin The residues determined

by peptide sequencing are underlined.

reticulum (SR) The specific effect of ablomin on high

K+-induced contraction therefore suggests that this

blockage was caused by the inhibition of voltage-gated

channels, rather than interaction with contraction-related

proteins such as ryanodine receptors, myosin, or

calmod-ulin that are found in the cytoplasm [47] In the rat-tail

artery, the intracellular Ca2+concentration is well

corre-lated with contraction force, and contraction evoked by

application of high extracellular K+ is completely

dependent on the influx of extracellular Ca2+ through

voltage-gated Ca2+channels [45,46,48] In this regard,

rat-tail arterial smooth muscle cells predominantly express

L-type Ca2+ channels among several subtypes of Ca2+

channels [49] For these reasons, we hypothesize that

ablomin may target voltage-gated Ca2+ channels on smooth muscle Further investigation is required to determine the target protein(s) In previous studies, HLTX

Fig 2 Isolation of ablomin from the venom of A blomhoffi (A)The venom of A blomhoffi was fractionated on a column of Superdex 75 pg (B) SDS/PAGE of continuous fractions eluted from Superdex 75 pg column under reduced condition The numbers above are elution volume (mL)of the fractions, and the arrows show ablomin The slightly larger protein, which eluted at 170–174 mL, was determined to be a serine protease-like venom protein by protein sequence analysis (C)Ablomin fractions (182–192 mL in elution volume)were subjected to a Q-Sepharose Fast Flow column and eluted with a linear gradient of NaCl (dotted line) Two-milliliter fractions were collected The fractions indicated by bar were pooled as ablomin (D)The ablomin fraction from c was subjected to a SP-Sepharose High Performance column and developed with a linear gradient of NaCl (dotted line) Two-milliliter fractions were collected The pooled fraction (bar) contained purified ablomin The result of SDS/PAGE of the purified ablomin is shown in inset (NR, nonreduced; R, reduced) Seven milligrams of ablomin were obtained from 1 g of crude venom For detailed purification procedures, see Experimental procedures.

Fig 3 Nucleotide and deduced amino-acid sequence of ablomin The

amino-acid sequence is shown in single-letter code beneath the

nuc-leotide sequence Nucnuc-leotide and amino acid (bold)number are shown

in the column at both sides Translation is depicted as starting at

nucleotide 66 The putative signal peptide is dotted underlined (from

)19 to )1 in amino acid number) The underlined shows the deduced

amino-acid sequence from enzymatic-digested S-pyridylethylated

peptides N-terminal was determined by the sequencing of intact

ablomin The putative poly adenylation signal is boxed.

has been shown to block ryanodine receptors on SR, in

addition to voltage-gated Ca2+ channels (including L-,

N-, and P-type)[43,44] In our current study, we used

intact arteries for measuring the activity of the protein,

which would presumably preclude access to cytoplasmic

proteins In the previous study, HLTX was applied to

purified SR membranes and membrane-permeabilized

ventricular trabeculae [43] Therefore, these experimental

differences are likely to account for the differences in

specificity and mechanism of action

Isolation and characterization of homologous proteins,

triflin and latisemin

In subsequent experiments, we isolated two other

homol-ogous proteins and cloned them by PCR Triflin (221

amino-acid residues, molecular mass 24 798 Da)was

puri-fied from the venom of the Habu snake (T flavoviridis) , and

latisemin (217 amino-acid residues, molecular mass

24 272 Da), was purified from the venom of the Erabu

sea snake (Laticauda semifasciata)(Figs 4 and 6A) In

typical purification procedures (see Experimental

proce-dures), we obtained 4 mg of triflin and 2 mg of latisemin

from 500 mg of crude venom, respectively The predicted

amino-acid sequences of triflin and latisemin are homolog-ous to that of ablomin (83.7 and 61.5%, respectively) Like ablomin, these proteins were capable of blocking contrac-tion of the artery induced by high K+(Fig 6B) These findings indicate that proteins homologous to ablomin are found in several snake venoms and represent new snake venom proteins with neurotoxin-like activities

D I S C U S S I O N

Snake venom neurotoxins, represented by a-neurotoxins and dendrotoxins, are thought to be found mostly in Elapidae snake venoms [21,31], and only a few snake venom neurotoxins have been isolated from Viperidae snakes [32–36] Recently, Brown et al isolated a 24-kDa cyclic nucleotide-gated ion channel blocker (designated pseudech-etoxin; PsTx)from the venom of the Australian King Brown snake (Pseudechis australis, Elapidae)[50] The N-terminal amino-acid sequence of PsTx has some identity

to those of the proteins in this study, although the complete amino-acid sequence of PsTx has not yet been reported (Fig 4) These facts strongly imply that other proteins that are homologous to ablomin may possess distinct biological activities In this regard, tigrin, which did not affect smooth

Fig 4 Sequence alignment of ablomin and structurally related proteins The residues conserved between the related proteins are shadowed Gaps (–) have been inserted to maximize similarity All cysteine residues are shown with black shading; gray shading shows identity The number of residues corresponds to that of ablomin The underline in ablomin, triflin, latisemin, and tigrin shows the amino-acid residues determined by peptide sequencing HLTX; helothermine, PsTx; pseudechetoxin, AEG; rat acidic epididymal glycoprotein (protein D/E), Ag5; hornet antigen 5, GliPR; human glioma pathogenesis-related protein, P14a; tomato pathogenesis-related protein P14a GenBank accession numbers, ablomin; AF384218, triflin; AF384219, latisemin; AF384220, tigrin; AY093955, HLTX; U13619, AEG; M31173, Ag5; Q05108, GliPR; JC4131, P14a; P04284 Note that the complete sequence of PsTx has not been published [50].

muscle contraction assay (Fig 6B), possibly possesses other biological activity, such as other neurotoxin-like activity The differences between structure and activity of these proteins led us propose possible interaction site(s), although

we could not confirm any potential active residue(s) The effective concentration for smooth muscle contrac-tion force herein is in almost the same range (Kiof 0.21 lM for ablomin obtained from double-reciprocal plots)as that

of a snake venom L-type Ca2+channel blocker calciseptine (IC50 ¼ 0.23 lM on rat aorta depolarization-induced contraction)[37] However, the blockage was not complete even at the concentration of 1 lM (Fig 5B) Higher concentrations of ablomin exposure (3 lM)did not induce further inhibition, but rather reduced the extent of inhibi-tion (Fig 5B) This decrease in the inhibiinhibi-tion of contracinhibi-tion

at higher concentrations was also found with 3 lMtriflin treatment In contrast, treatment with dihydropyridines, the definitive blockers of L-type Ca2+channels, decreased the arterial smooth muscle contraction to < 20% of the con-trol Because of the structural similarity with HLTX, it is possible that ablomin and two homologous proteins target multiple ion channels The possible multiplicity of targets may underlie the partial inhibition seen in our experiments, but further experiments are needed to fully elucidate the factors that cause partial inhibition

The structural alignment appears to show that these proteins are classified into the CAP family of proteins (CRISPs, Antigen 5 proteins, Pathogenesis-related pro-teins) The primary structural characteristic of the CRISPs

is a high content of cysteine residues [16] Although the functions of most of the CRISP family proteins are unknown, some are thought to play roles in the immune system and sperm maturation [51,52] Antigen 5 proteins are components of the venoms of wasps and ants, and they are thought to be involved in the provocation of an acute and localized inflammatory response [53] The pathogenesis-related proteins of plants, e.g tomato P14a in Fig 4, are produced during the defence reaction of plants against pathogenic infection and environmental stress [54] How-ever, no distinct target molecules have yet been proposed for these family proteins Although the identity of the CAP proteins vs ablomin are relatively low, i.e 42.7% for acidic epididymal glycoprotein, 26.9% for antigen 5, 27.0% for glioma pathogenesis-related protein, and 29.5% for P14a (Fig 4), ablomin and homologous proteins possess a highly conserved GHF(Y)TQI(V/M)VW sequence among the CAP family proteins at position 114–121 Recently, the NMR structure of tomato pathogenesis-related protein P14a was reported and putative active sites of pathogenesis-related proteins (two histidines and glutamates)have been proposed [55,56] These residues are also completely con-served in the proteins, which we isolated (His60, Glu75, Glu96, and His115 in ablomin in Fig 4) These facts strongly suggest these proteins have a three-dimensional structural similarity with other CAP family proteins, indi-cating their possibility as models for elucidating the virtually unknown functions of CRISP (CAP)family proteins

In conclusion, we have isolated several novel snake venom proteins One of these proteins, ablomin, which was purified from the venom of A blomhoffi, blocks high K+ -induced contraction of arterial smooth muscle We also cloned the cDNA encoding ablomin from the venom gland

of A blomhoffi and determined its complete sequence

Fig 5 Ablomin specifically inhibits high K + -stimulated contraction of

rat-tail arterial smooth muscle (A)Blockage of high K+-induced

contraction by ablomin Smooth muscle strips were pretreated with

1 l M ablomin, then exposed to stimulants (60 m M K + or 20 m M

caffeine) To rule out the possible effect of norepinephrine, the H-T

solutions contained 1 l M prazosin to block a1 adrenergic receptors.

(B)Concentration dependency of ablomin on high K + - or

caffeine-stimulated contraction by ablomin (n ¼ 4, mean ± SEM, asterisk,

P < 0.05).

Fig 6 Several ablomin-like snake venom proteins block high K +

-induced contraction in smooth muscle (A)SDS/PAGE of snake venom

proteins homologous to ablomin under nonreducing (lane 1–4)and

reducing (lane 5–8)conditions The positions of molecular mass

markers are shown on the left Lanes 1 and 5 show ablomin (26 and

29.7 kDa), lanes 2 and 6 show triflin (23 and 29 kDa), lanes 3 and 7

show latisemin (28 kDa each), and lanes 4 and 8 show tigrin (28 and

30 kDa) The numbers on the left are relative molecular mass of

standard molecular marker proteins (B)The effect of ablomin

ho-mologous proteins on high K+-induced smooth muscle contraction.

Smooth muscle strips were pretreated with each protein at 1 l M , then

stimulated with 60 m M K + as described in Fig 5 (n ¼ 4, mean ±

SEM, asterisk, P < 0.05).

Furthermore, we demonstrated that ablomin homologous

proteins with similar neurotoxin-like activities, named triflin

(T flavoviridis)and latisemin (L semifasciata), are

distri-buted in other Viperidae and Elapidae snake venoms To

date, HLTX is the only neurotoxin that has been classified

into the CRISP family of proteins so far Our present results

strongly suggest that HLTX-like proteins are widely

distri-buted in several snake venoms

A C K N O W L E D G E M E N T S

We thank Dr Sadaaki Iwanaga for providing the lyophilized venom of

A blomhoffi, and Satsuki Hori for technical assistance We would also

like to thank Dr R Lane Brown (Oregon Health & Science University,

OR, USA)for helping to revise the language of the manuscript This

work was supported in part by Scientific Research Grants-in-Aid from

the Ministry of Education, Science and Culture of Japan (to T M).

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